Use of L-Carnitine as stabilizing agent of proteins

ABSTRACT

The present invention relates to the technical field of stabilizing proteins, in particular to the therapeutic aspects of protein stabilization. L-carnitine is a useful agent for stabilizing proteins, and in a particularly favourable aspect in proteins used in the medical field. In a preferred aspect, L-carnitine is used for protecting chaperone activity, and in the medical field for preserving the activity of altered chaperone proteins. In connection with this invention L-carnitine is used for the preparation of a medicament for the treatment of diseases due to altered chaperone proteins, such as eye diseases, in particular cataract.

This application is the US national phase of international applicationPCT/IT00/00520 filed 15 Dec. 2000 which designated the U.S.

The present invention relates to the technical field of stabilizingproteins, in particular to the therapeutic aspects of proteinstabilization.

BACKGROUND OF THE INVENTION

In the production of proteins and polypeptides, either by extraction orby recombinant biotechnology techniques, there is the problem ofmaintaining the correct folding of the protein so as to keep its desiredactivity.

Unfolding or incorrect or otherwise modified foldings may occur becauseof technical manipulation or the general processing system for theproduction of the proteins.

Another problem in proteinaceus material processing is given by theaggregation of proteins.

Many solutions are offered in the state of the art. Some of them arepeculiarly chemical, meaning by this that chemical reagents are used,such as, for example particular mixtures of salts, even in buffersolutions.

U.S. Pat. No. 5,728,804, to research Corporation Technologies, disclosesa method for protein renaturation by means of detergent-freecyclodextrins. U.S. Pat. No. 5,563,057, to Wisconsin Alumni ResearchFoundation, other than cyclodextrin, teaches the use of certaindetergents for refolding misfolded enzymes.

U.S. Pat. No. 5,874,075, to Amgen, discloses protein:phospholipidscomplexes useful for stabilizing proteins against thermally-inducedaggregation, denaturation and loss of activity.

U.S. Pat. No. 5,756,672, to Genentech, provides a composition comprisinga polypeptide in a certain buffer. Said buffer being suitable forrefolding improperly folded polypeptides. A particular embodiment isgiven for refolding misfolded insulin-like growth factor-I.

The above mentioned methods might be convenient, since easily availablechemicals are used, but may raise some instances for certain chemicalsused, for example copper or manganese salts (U.S. Pat. No. 5,756,672).

Refolding occurs also through chromatographic techniques, See AltamiranoM M. Et al. Nat. Biotechnol. 1999 February; 17(2):187-91.

The discovery of chaperonins has opened a new field for the technologyof protein processing.

Chaperonins, also known as heat-shock proteins or HSP; are naturalproteins exerting a biological role in protein folding. See for anextensive review internet address ermm.cbcu.cam.ac.uk/000021015h.htm byJulia C. Ranford, Anthony R. M. Coates and Brian Handerson.

Technically, chaperonins are intensively studied as means for facing theabove-mentioned problem of protein stabilization and refolding.

This search leads to newly discovered chaperonins and to their use forprotein stabilization, see for example U.S. Pat. No. 5,428,131, to YaleUniversity. For a picture of chaperonins for the technical problem facedby the present invention, see for example U.S. Pat. No. 5,688,651, toRAMOT University; U.S. Pat. No. 5,646,249, to U.S. Health Department;U.S. Pat. No. 5,561,221, to Nippon Oil Company Limited, WO 00/20606, toReiman and Schirmbeck, J P 11266865, to Kaiyo Biotechnology Kenkyusho KK, WO 99/40435, to Netzer; JP 10327869, to Kaiyo Biotechnology KenkyushoK K; WO 00/71723, to Roche Diagnostics; WO 00/55183 and WO 99/05163, toMedical Research Council.

Chaperonins are a useful tool for protein stabilization and refolding,but some technical drawbacks come from their use. Since they areproteins, even they are prone to alteration, such as thermal one, sothey too need some protective factor.

For the technical field of stabilizing proteins, this problem is alsovery important for preparation of HSP cancer vaccines. It has beenobserved that the immunogenicity of a given antigen is rendered far moreefficient when it is presented to immune cells in a complex with HSPs(Requena J M et al, Ars-Pharm 1997, 38(2-3):191-208; Castellino F, etal, J Exp Med 2000, 191(11):1957-1964). Particularly, the immuneresponse to cancer is boosted with HSP (i.e., HSP70 or gp96) which arelinked to an antigenic peptide (“specific antigenic fingerprints”), bothof which are obtained from the patient's cancer cells (Yedavelli Spet alInt J Mol Med 1999, 4(3):243-248). It is therefore important to havestable and/or well preserved HSP for cancer vaccines.

Interestingly, some chaperoning, such as the eye lens alpha-crystallinproteins, are members of the small heat shock protein (sHSP) family.sHSPs have been shown to function in a number of different processesranging from RNA stabilization to elastase inhibition and interactionwith the cytoskeleton.

AlphaA-crystallin is localized primarily in the lens with very lowlevels found in other tissues, whereas alphaB-crystallin is now known tobe essentially ubiquitous throughout the body (Haley D A et al, J MolBiol 1998, 277:27-35). The biological importance of alphaB-crystallin ishighlighted by its elevated levels in ischemic heart and in the brainsof patients with multiple sclerosis, Alzheimer's and other neurologicaldiseases. Consistent with its classification as an HSP, expression ofalphaB-crystallin has been shown to be induced by a variety ofphysiological stresses including heat, osmotic stress, and metaltoxicity. The biological importance of alphaA-crystallin in lens ishighlighted by its efficient suppression of uncontrolled aggregation ofdamaged proteins.

As it appears from the above examples, the stabilization of chaperoning,either for their use or for stabilizing them in those pathologicalstates in which their are altered, is very important in the medicalfield.

ABSTRACT OF THE INVENTION

It has now been found that L-carnitine has a surprising effect instabilizing proteins, and in a particularly favourable aspect for themedical field, L-carnitine has a surprising effect in protectingchaperone activity, this protecting activity being exerted through astabilizing effect by L-carnitine toward chaperone activity.

Therefore, it is an object of the present invention the use ofL-carnitine or of a salt thereof for stabilizing proteins, in particularas auxiliary factor in protecting chaperone activity.

In one preferred embodiment of the present invention, L-carnitine isused in the therapeutical field, for preserving the activity of alteredchaperone proteins.

In connection with the first preferred embodiment, L-carnitine is usedfor the preparation of a medicament for the treatment of diseases due toaltered chaperone proteins.

In a second preferred embodiment of the present invention, L-carnitineis used for the preparation of a medicament for the treatment ofdiseases based on altered activity of alpha-crystallin protein, moreparticularly AlphaA- and/or alphaB-crystallin. Diseases that may betreated are for example ischemic heart and in the brains of patientswith multiple sclerosis, Alzheimer's and other neurological diseases. Ina third preferred embodiment, the disease is an ophthalmic affectionwhere α-crystallin is the altered chaperone protein. In a particularaspect, the present invention applies in the treatment of cataract,excluding cataract of diabetic origin.

Proteins, in particular chaperonines, such as HSP, stabilized by meansof L-carnitine are also an object of the present invention.

Accordingly, further objects of the present invention are the use ofL-carnitine or a pharmaceutically salt thereof for the manufacture of amedicament for the treatment of cataract of non-diabetic origin.

A particular object of the present invention is an ophthalmiccomposition comprising a suitable amount of L-carnitine or apharmaceutically salt thereof. This ophthalmic composition canoptionally comprise a further active ingredient useful for the treatmentof cataract of non-diabetic origin.

The above objects of the present invention shall be illustrated indetail also by means of examples.

DETAILED DESCRIPTION OF THE INVENTION

For pharmaceutically acceptable salt of L-carnitine, it is intended anysalt, organic and inorganic suitable for use in medical field, human andveterinary. In the general field of protein stabilization, any salt maybe used, with the condition that it is compatible with the specificapplication.

Examples of pharmacologically acceptable salts of L-carnitine, thoughnot exclusively these, are chloride; bromide; iodide; aspartate; acidaspartate; citrate; acid citrate; tartrate; acid tartrate; phosphate;acid phosphate; fumarate; acid fumarate; glycerophosphate; glucosephosphate; lactate; maleate; acid maleate; mucate; orotate, oxalate;acid oxalate; sulphate; acid sulphate; trichloroacetate;trifluoroacetate; methane sulphonate; pamoate and acid pamoate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The ocular lens is a transparent organ constituted by a highlyconcentrated and highly ordered matrix of structural proteins, called‘crystallins’, which are probably the longest lived proteins of the body(Wistow, G., and Piatigorsky, J. (1988), Ann. Rev. Biochem, 57, 479-504;Blomendal, H: (1982) Biochem. 12, 1-38; Bettelheim, F. A. (1985) TheOcular Lens. Structure, Function, and Pathology, (Maisel H. ed.) pp.265-300, Marcel Dekker, Inc, New York; Tardieu, A., and Delay, M.(1988), Ann. Rev. Biophys. Chem. 17, 47-70).

Post-translational modifications of lens crystallin, consequent to agingor diseases such as diabetes, may result in conformational changes andaggregation of these proteins, and lead to lens opacification andcataract formation (Harding, D. (1981), Molecular and Cellular Biologyof the Eye Lens (Bloemendal H., ed) pp. 327-365, John Wiley and Sons,New York). Although the mechanisms of cataractogenesis are not wellunderstood, oxidation of lens proteins is associated with cataract inmammals (Francis, P. J. (1999), Trends in Genetics 15, 191-196).

The lens undergoes major oxidative stress because it is constantlyexposed to light and oxidants (Varma, S. D., et al. (1984), Curr Eye Res3, 35-57, Spector, A. (1995), FASEB J. 9, 1173-1182; Taylor, A., andDavies, K J. (1987), Free Radic Biol Med 3, 371-377; Zigman, S. (1981),Mechanisms of Cataract Formation in the Human Lens (Ducan G, ed.) pp.117-149, Academic press, New York; I. Dillon, J. (1985), The OcularLens. Structure, function, and Pathology, Maisel H. ed, Marcel Dekker,Inc., New York, 349-366 Zigman, S. (1985), The Ocular Lens. Structure,Function and Pathology. (Maisel H. ed.) pp. 301-347 Marcel Dekker, Inc.,New York). Oxidative modifications include selective oxidation ofspecific amino acids that results in charge alterations, proteindegradation, protein cross-linking and insolubilization, and increasednon-tryptophan fluorescence (Spector, A., and Gamer, W. H. (1981), ExpEye Res. 33, 673-681 Andley, U. P. (1987), Photochem. Photobiol. 46,1057-1066 Davies, K. J. A., et al (1987), J Biol Chem. 262, 9914-9920Augusteyn, R. C. (1981), Mechanisms of Cataract Formation in the HumanLens (Ducan, G., ed.) pp. 72-115, Academic Press, London Zigler, J. S.Jr et al (1989), Free Radic Biol Med. 7, 499-505). Consequently, thelens has developed antioxidant systems and repair mechanisms tocounteract the effect of oxidants. The first line of defense againstoxidation stress is constituted by radical scavenging antioxidants thatreduce the oxidative insult. For example, glutathione (GSH) and taurine,which are both highly represented in lens tissue, exert protectiveeffects in an in vitro model of diabetic cataract (Richard R C Et al(1998), Proc Soc Exp Biol Med 217, 3 97-407 Jones, R. A. V. andHothersall, J. S. (1999), Exp Eye Res. 69, 291-300). Furthermore,α-crystallin, which constitutes up to 50% of the total protein mass ofthe mammalian lens, acts as a molecular chaperone that preventsheat-induced aggregation of numerous proteins and is required for therenaturation of chemically denaturated proteins (Jones, R. A. V. andHothersall, J. S. (1999), Exp Eye Res. 69, 291-300). A key element ofα-crystallin function is its ability to prevent aberrant proteinassociations by binding to transiently exposed hydrophobic proteinsurfaces (van den Ussel P. R. et al. (1996), Ophthalmic Res. 28, 39-43).Because α-crystallin prevents both ultraviolet radiation- and freeradical-induced aggregation of proteins in vitro (Groenen P J et al(1994) Eur. J Biochem. 225, 1-19 Andley, U. P. et al (1998) J Biol Chem.273, 31252-31261; Lee, J. S. et al (1997) J Protein Chem. 16, 283-289;Kramps, J. A. Et al (1978), Biochem Biophys Acta 533, 487-495; VanKleef, F. S. M., et al (1976), Eur J Biochem 66, 477-483 Smulders, R. H.P. H. et al (1996), J Biol Chem 271, 29060-29066.), it may also protectlens proteins from photooxidative changes in vivo.

U.S. Pat. No. 5,037,851, issued on Aug. 6, 1991, with a priority claimof Nov. 15, 1988, in the name of the same assignee of the presentinvention, claims a therapeutic method for the treatment of cataractwhich comprises administering orally or parenterally to a subject havinga cataract 1000 to 2000 mg/day of acetyl D-carnitine or an equivalentquantity of one of its pharmacologically acceptable salts. The teachingof this patent is limited to acetyl D-carnitine.

The present inventor and colleagues previously showed that inexperimental animal diabetes the decrease in lens carnitine, aubiquitous molecule involved in many biological pathways, is an earlyimportant and selective event possibly related to cataract formation(Pessotto P, et al. (1997) Exp. Eye Res. 64, 195-201). In this paper,there is disclosed how in the diabetic states there is a loss ofL-carnitine in the lens and the carnitine levels in the other eyetissues seem substantially unaffected. The authors conclude that therole of L-carnitine in lens is still unclear, but its loss may berelated to the appearance of cataract. There is a strong suggestion inthis reference to use acetyl carnitine, giving a good reason forexpectation of success, for the prevention of the appearance of cataractby a pharmacological action, as has been shown for aspirin. The reasonfor an expectation of a favourable action by acetyl carnitine, in viewof the well-known action of aspirin, is that both compounds haveacetylating properties, which, on their turn are responsible for theprotection of lens proteins.

In addition to its primary function as a carrier of long-chain fattyacids from the cytoplasm to the sites of β-oxidation, it has beenanticipated that L-carnitine could also serve to maintain cellhomeostasis.

Swamy-Mruthinti, S., and Carter, A. L. (1999), Exp Eye Res 69, 109-115demonstrate that L-carnitine results ineffective in in vitro glycationof lens crystallins, while acetyl L-carnitine and acetyl salicylic aciddecreased crystallin glycation. These considerations explain whyL-carnitine levels in various animal tissues do not invariably correlatewith tissue energy requirements or with lipid metabolism. For example,the eye lens, a non-vascularized tissue whose main source of energy isglucose absorbed from ocular fluids, has higher L-carnitineconcentrations than other eye compartments (Pessotto, P. et al (1994) JOcul. Pharmacol. 10, 643-651).

1. This invention accordingly contemplates the use of L-carnitine andits pharmacologically acceptable salts to produce an ophthalmicpharmaceutical composition for the therapeutic treatment of cataract, inparticular cataract of non-diabetic origin. In practice, atherapeutically effective amount of L-carnitine or an equivalentquantity of one of its pharmacologically acceptable salts isadministered to the eye, optionally comprising a further activeingredient useful for the treatment of cataract of non diabetic origin.

Preferably, the composition is in the form of a collyrium. The collyriumis applied to the extent of the therapeutic necessity, as determined bythe skilled person and depending on the conditions of the patient, theseverity of the illness and any other factor considered by the skilledperson. For example, 2-3 drops 3-4 times daily may be suitable.

The compositions for the collyrium comprise the usual sterile isotonicsolution. The choice of the suitable excipients is within thecapabilities of a normally skilled person in pharmaceutical technology.For example, use is made of excipients such as sodium chloride, dibasicsodium phosphate, monobasic potassium phosphate, benzalkonium chloride,and ethyl alcohol. The composition is brought to the correct volume withdistilled water.

EXAMPLE Materials and Methods

Lens Organ Culture

Four-month-old Sprague-Dawley rats were anesthesized with 5 mg/kgxylazine and 65 mg/kg ketamine, and decapitated. Immediately after, eyeswere enucleated, extracted and placed in 2 ml of modified TC-199 medium.Lens integrity was assessed by measuring protein leached into the mediumafter 30-60 min of culture; damaged lenses were discarded. One lens ofeach pair was placed in culture medium with no H₂O₂ and used as acontrol. After 24 h of culture, the control lenses did not differ fromfreshly enucleated lenses in any of the parameters evaluated in thisstudy. The controlateral lens of each pair was placed in medium and,after equilibration under 5% CO₂ at 37° C., was exposed to 500 μM H₂O₂in the absence or presence of 300 μM L-carnitine. After 24 h ofincubation, morphological characteristics and changes were recorded, andthe lenses were photographed. The incubated lenses were rinsed withsaline solution, blotted on filter paper, weighed, and then immediatelyprocessed for biochemical analysis. To determine lactate dehydrogenase(LDH) leakage, lenses were incubated individually in each differentmedium, and the medium was harvested daily and saved for LDH analysis.

Extraction of Lens Proteins

Decapsulated lenses were homogenized with disposable pestles and thensonicated in extract buffer (20 mM HEPES, 0.2 mM EDTA, 0.5 mMdithiothreitol, 450 mM NaCl, 25% glycerol, 0.5 μM/ml leupeptin, 0.5μg/ml protinin, 0.5 mM phenylmethanesulfonyl fluoride) on ice. Aliquotsof the homogenate from each of the incubated lenses were removed for GSHand L-carnitine analysis. The remainder was centrifuged for 25 min at20,000×g to separate the supernatant from the pellet. The pellet waswashed with 1.0 ml buffer and dried under nitrogen. This fraction wasdesignated “water-insoluble fraction”. The supernatant fraction wasdialyzed twice against 3 ml of 0.025 mol/l phosphate buffer, pH 7.4 for48 h and lyophilized. This fraction was designated “water-soluble”fraction. The water-soluble and water-insoluble fractions weredelipidated with 3.0 ml chloroform:methanol (2:1) for 16 h under shakingfollowed by centrifugation at 2,000 g for 5 min.

After the organic solvent was discarded, the residue was treated with2.0 ml of diethyl ether, left to stand for 5 min and then centrifuged at2,000 g for 5 min. The pellet was dried under air and stored at 4° C. ina desiccator.

Preparation of Crystallins

The water-soluble crystallin fractions were isolated by preparativeSephacryl S-300-HR gel permeation chromatography as previously described(Smulders, R. H. P. H. et al (1996), J Biol Chem 271, 29060-29066., deJong, et al (1974), Eur J Biochem. 48, 271-276). Briefly, solubleprotein was applied to a 100×1.5-cm column and developed isocraticallywith phosphate buffer. The total fractions from control andH₂O₂±L-carnitine treated lenses were concentrated by ultrafiltration ina Diaflo apparatus and their purity checked by SDS-PAGE, done accordingto Laemmli using a Bio-Rad Mini-Protean II System. Proteinconcentrations were measured with a Bradford protein assay kit(Bio-Rad).

Western Blotting

Total lens homogenate was applied on 4-20% gradient sodium dodecylsulphate (SDS) gels using. Tricine buffers and then transferred topolyvinylidene difluoride membranes. Western blotting was performed asdescribed elsewhere (Kim, S. Y. et al (1995), J Invest Dermatol 104,211-217). The concentration of antibodies was 5 μg/ml for primaryantibody (anti-γ-glutamyl-ε-lysine isopeptide) and 0.1 μg/ml forsecondary antibody. The blot was then developed by enhancedchemiluminescence (Pierce, Milan, Italy). Subsequently, the very highmolecular weight bands were cut out, eluted into SDS buffer containingTricine, freed of SDS by ion pair extraction (Konigsberg, W. H., andHenderson, L. (1983) Proc Natl Acad Sci USA. 80, 2467-2471), andsubjected to amino acid analysis.

Measurement of isopeptide cross-links in water-insoluble proteins.

The water-insoluble proteins were suspended in 0.2 M N-ethylmorpholineacetate (pH 8.1). An aliquot (10%) was used to quantitate the amount oftotal protein. Samples were digested by the sequential addition ofproteolytic enzymes (collagenase, pronase, aminopeptidase andcarboxypeptidase A, carboxypeptidase B and carboxypeptidase y), directlyto the reaction mixture at 37° C. in the presence of 0.02% sodium azide.After enzymatic digestion, the free N-(γ-glutamyl)lysine isopeptidecross-link was resolved by HPLC and quantitated by amino acid analysis(Hohl, D., et al (1991), J Biol Chem. 266, 6626-6636). In a related setof experiments, the isopeptide content of lenses was determined withoutprior extraction.

Tryptophan Fluorescence

The loss of-protein tryptophan fluorescence, an indicator of tryptophanoxidation, seems to be a marker of crystallin integrity. We thereforemeasured-tryptophan fluorescence in lens crystallin (Perkin-Elmer 650-40spectrophotometer) according to a previously described method (Jones, R.H. V., and Hothersall, J. S. (1993), Exp Eye Res 57, 783-790). Theexcitation wavelength was set to 295 nm, and the fluorescence emissionwas detected at 330 nm.

Evaluation of the molecular chaperone activity of α-crystallins fromcontrol and in vitro-treated rat lenses

The following experiments were performed essentially as describedelsewhere (Horwitz, J. (1992) Proc Natl Acad Sci USA. 89, 10449-10453).The chaperone-like activity of α-crystallin from control andH₂O₂±L-carnitine treated lenses was determined by heat denaturationstudies. The extent to which the unmodified or modified α-crystallinpreparation protected β_(L)-crystallin (used as the target protein) fromheat-induced denaturation and aggregation was assessed as follows: 100μg or 200 μg of α-crystallin were added to 200 μg of β_(L)-crystallin ina 1.5-ml cuvette and made up to a final volume of 1 ml with 50 mMphosphate buffer, pH 7.0. The cuvette was placed in atemperature-regulated cell holder attached to a UV spectrophotometer.Light scattering due to protein denaturation and aggregation wasmonitored at. 360 nm absorbance for 3,000 s at 55° C. or for 1,800 s at58° C.

Intermediate filament assembly and viscosity assays involvingα-crystallins

The sedimentation assay devised by Nicholl and Quinlan (Nicholl, I. D.,and Quinlan, R. A. (1994), EMBO J 13, 945-953) was used to assessα-crystallin-induced inhibition of intermediate filament assembly.Purified porcine glial fibrillary acidic: protein (GFAP) was used forthese studies; it was purified from porcine spinal cord by axonalflotation as described previously (Pemg, M. D., et al (1999), J CellSci. 112, 2099-2112, MacLean-Fletcher, S., and Pollard, T. D. (1980),Biochem Biophys Res Commun 96, 18-27). The gel formation assay was basedon a method used to monitor actin binding protein activity by fallingball viscometry (Pemg, M. D., et al (1999), J Cell Sci. 112, 2099-2112).α-Crystallins were mixed with GFAP in 8 M urea, 20 mM Tris-HCI, pH 8.0,5 mM EDTA, 25 mM 2-mercaptoethanol and then stepwise dialyzed in 10 mMTris-HCI, pH 8.0, 25 mM 2-mercaptoethanol. Assembly of the GFAPintermediate filaments, in the presence or absence of α-crystallin, wasinduced by the addition of a 20-fold concentrated buffer to give a finalconcentration of 100 mM imidazole-HCI, pH 6.8, 0.5 mM DTT. A 100-μlaliquot of sample was loaded into a glass tube and used for theviscosity assay. The tube was then immersed in a 37° C. water bath for 1h before the gel formation assay. A ball was then placed into the tube,and the ability of the solution to support the ball was monitored.

Lens Microscopic Examination

After a 24-h incubation with or without H₂O₂ in the presence or absenceof L-carnitine, lenses were submitted to standard procedures forhistologic analysis. For optical microscopy, lenses were removed fromculture medium, immersed in fixative (neutral buffered formalin),dehydrated in ethanol, cleared in xylene, and embedded in paraffin waxfor sectioning. Five micrometer sections were prepared and stained withhematoxylin and eosin. For scansion electron microscopy the lenses werefixed by immersion, for at least 24 h at room temperature, in a solutionof 2.5% glutaraldehyde and 6% sucrose, buffered to pH 7.2 with 50 mMsodium cacodylate. Samples were dehydrated through a graded series ofethanol, critical point-dried using CO₂, mounted on aluminium stubs,sputter-coated with gold, and examined with a Leica Stereoscan 440microscope at a 3-7 kV acceleration voltage.

For transmission electron microscopy, the lenses were fixed as describedabove for the scansion electron microscopy procedure, postfixed in OsO₄buffered with 150 mM sodium-potassium phosphate (pH 7.4), embedded,sectioned, and stained for electron microscopy. They were examined at aJEOL 100B electron microscope.

Results

Changes in Lens Morphology

After 24 h of incubation, the lenses incubated without H₂O₂ (controllenses) retained their clarity, but those exposed to 500 mM H₂O₂ becameuniformly cloudy throughout the outer cortical region and were swollen(data not shown). As shown in Table 1, at the end of incubation,H₂O₂-treated lenses were significantly heavier than control lenses(47±0.2 mg vs 25±0.1 mg). There were no differences in weight betweencontrol lenses and lenses treated with both L-carnitine and H₂O₂. Thelenses treated with H₂O₂ alone became opaque, whereas lenses treatedwith L-carnitine and H₂O₂ remained clear. Optical and electronmicroscopy showed that cell shape was unaltered and that fiber cellswere intact in control lenses and in lenses treated with L-carnitine andH₂O₂. Ballooning, liquefaction and various degrees of fiber swellingwere observed in lenses exposed to H₂O₂ alone.

L-carnitine and GSH concentrations in control and H₂O₂-treated lenses

Under our experimental conditions, there was no significant differencein GSH and L-carnitine concentrations in control lenses, whereastreatment with 500 mM H₂O₂ caused a precipitous drop in GSH andL-carnitine levels (Table 1). The addition of L-carnitine (300 mM) tothe lens incubation medium before H₂O₂ treatment did not prevent theloss of GSH, but maintained the carnitine concentration almost at thelevel found in control lenses. To determine whether the decrease of GSHand L-carnitine was related to lens damage, we measured leakage of LDHinto the medium. As expected, after H₂O₂ treatment the decrease in GSHand L-carnitine levels was accompanied by a significant increase of LDHin the supernatants, indicating that depletion of these factors wasindeed associated with lens damage. To determine the protective, effectof L-carnitine, we incubated lenses in culture medium containing 300 mMof the molecule. As expected, the concentration of GSH in lenses treatedwith L-carnitine and H₂O₂ decreased to about the same level as in lensesexposed to H₂O₂ alone, but the concentration of LDH in the medium fromthe lens treated with L-carnitine and H₂O₂ was similar to that observedin control lenses. This indicates that L-carnitine can withstand thisconcentration of H₂O₂.

Recovery of high molecular weight proteins in the water-insoluble lensfractions containing isopeptide cross-links.

Water-insoluble proteins constituted only 5% of total proteins incontrol lenses, but increased to 41% of total proteins in H₂O₂-treatedlenses (Table 1). The concentrations of water-insoluble proteins inlenses treated with L-carnitine and H₂O₂ were the same as those observedin control lenses.

Chaperone-like Function of α-crystallin

The chaperone properties of the purified water-soluble α-crystallin weredetermined by crystallin (target protein) aggregation assay.Characteristically, β_(L)-crystallin aggregates at elevatedtemperatures. The addition of α-crystallin either prevents or decreasesthe heat-induced aggregation of β_(L)-crystallin, which is measured bylight scattering at 360 nm. Since the ratio of α to β determines thedegree of protection against heat-induced aggregation, we used 100 μg or200 μg of α-crystallin and 200 μg of β_(L)-crystallin. As expected,α-crystallin from control lenses exerted chaperone activity.After—incubation with H₂O₂, there was a significant decrease in thecapacity of —α-crystallin to prevent the heat-induced aggregation ofβ_(L)-crystallin, whereas the presence of L-carnitine in the lensincubation mixture prevented this negative effect.

Gel Forming Assay

Since intermediate filaments such as GFAP are a physiological target ofα-crystallins, we tested α-crystallin chaperone activity using fallingball viscosimetry in the gel forming assay (MacLean-Fletcher, S., andPollard, T. D. (1980), Biochem Biophys Res Commun 96, 18-27).

GFAP is an appropriate target because of the property of α-crystallin todisaggregate GFAP cytoplasmic inclusions. In the absence ofα-crystallin, GFAP forms a protein gel that supports the ball used inthe viscosity test. To determine whether H₂O₂ treatment affected thecapacity of lens α-crystallin to disrupt the GFAP network, α-crystallinfrom control or from H₂O₂±L-carnitine-treated lens was added to the gelforming assay. α-Crystallin from control lenses completely inhibited theformation and maintenance of the GFAP gel in the viscosity assay,whereas α-crystallin from lenses treated with H₂O₂ alone did not affectgel formation. In addition, α-crystallin from lenses treated with bothL-carnitine and H₂O₂ blocked GFAP gel formation to the same extent asα-crystallin from control lenses.

Tryptophan Fluorescence Measurements

Tryptophan fluorescence was measured in α-crystallin fractions fromcontrol and treated lenses to identify conformational changes. Inα-crystallin from H₂O₂-treated lenses there was a 2.7-fold loss oftryptophan fluorescence; again, L-carnitine restored the basal value.

Lenses exposed to L-carnitine and oxidative stress remained transparent.Although the present inventors do not wish to be bound to any theory,the protective effect of L-carnitine is not easily explained becauseL-carnitine per se is not known to exert antioxidant activity (Arduini,A. et al (1992), J Biol Chem. 267, 12673-81). Neither did L-carnitinerescue GSH depletion, which means that the beneficial effect was notmediated by an increase of GSH through, for example, an anapleroticeffect on NADPH, a cofactor of the glutathione reductase enzyme (Pemg,M. D., (1999), J Biol. Chem 47, 3323 5-33243. Wuensch, S. A., and Ray,P. D. (1997), Comp Biochem Physiol B Biochem Mol Biol 118, 599-605).Rather, the fact that LDH leakage into the medium, was not increased inlens treated with L-carnitine and exposed to oxidative stress indicatesthat the molecule can sustain lens integrity. Here we show that lensα-crystallin chaperone activity is diminished by in vitro oxidativestress, and provide support for the proposal that lens proteinssubjected to oxidative insult sustain a high degree ofpost-translational modifications (Cherian, M., and Abraham, E. C.(1995), Biochem Biophys Res Commun. 212, 184-189). L-carnitine not onlyreduced the increased post-translational modifications of lens proteinsbut also afforded significant protection against the decreased chaperoneactivity of α-crystallin. α-Crystallin has been shown to suppressaggregation of denatured proteins in studies in which mixtures ofthermally stressed β-crystallins served as substrate (Kramps, J. A. Etal (1978), Biochem Biophys Acta 533, 487-495). It has been demonstratedthat oxidative stress disrupts α-crystallin chaperone activity, which iscrucial for maintenance of lens transparency (Zigler, J. S. Jr et al(1989), Free Radic Biol Med. 7, 499-505; Richard R C Et al (1998), ProcSoc Exp Biol Med 217, 3 97-407). Therefore, L-carnitine beneficiallyaffects lens transparency by acting directly on α-crystallin.

Both α- and β-crystallins are N-terminally acetylated. Using screeningspot-blot analysis combined with mass spectrometry, Takernoto et al.provided evidence that the N-acetylated-terminal methionine ofα-crystallin can be oxidized to methionine sulfoxide in vivo (Sims, N.R., et al (2000), Brain Res Mol Brain Res. 77, 176-184). This oxidationof the N-terminal methionine, which is exposed on the outside of thepolypeptide, can negatively affect the function of the protein. Inaddition to NH3-terminal acetylation, the -amino groups of lysine (Lys)residues are subject to acetylation. All seven Lys residues of bovine(α-crystallin react with aspirin, the extent of acetylation varying from10% for Lys 88, the least reactive, to 60% for Lys 166, the mostreactive (Takemoto, L. et al (1992), Curr. Eye Res. 11, 651-655; Rao, G.N. et al (1985), Biochem. Biophys. Res. Commun. 128, 1125-1127). Aspirininhibits both glycation and carbamoylation as well as aggregation oflens proteins, presumably through acetylation of Lys residues (Hasan, A.et al (1993), Exp Eye Res. 57, 29-35; Cromptonm, Rixon, K. C., andHarding, J. J. (1985), Exp. Eye Res. 40, 297-311; Rao. G. N., andCotlier, E. (1988) Biochem. Biophys. Res. Commun. 151, 991-996; Huby,R., and Harding, J. J. (1988), Exp Eye Res. 47, 53-59; Ajiboye, R., andHarding D. (1989), Exp Eye Res. 49, 31-41; Blakytin, R., and Harding J.J. (1992), Exp Eye Res. 54, 509-518). Recently, it has been shown thatacetyl-L-carnitine inhibits glycation of (α-crystallin, to a greaterextent than other crystallins, through acetylation of the potentialglycation sites (Groenen, P. J. et al (1993), Biochem J. 295, 399-404).Glycation seems to be more harmful than acetylation because onlyglycation products are involved in protein cross-linking and in asignificant decrease of the α-crystallin chaperone activity (Groenen, P.J. et al (1993), Biochem J. 295, 399-404, Blakytin, R., and Harding J.J. (1992), Exp Eye Res. 54, 509-518).

TABLE 1 Changes of some biochemical parameters in control lenses and inH₂O₂ ± L-carnitine-treated lenses. Water Free Acetyl Total Lensinsoluble Glutathione carnitine carnitine LDH (units/ml carnitine weightprotein (μmoles/g (nmoles/g (nmoles/g conditioned (nmoles/g (mg) (%)w-w) w-w) w-w) media) w-w) Controls 25 ± 0.1 5 ± 1.1 4.87 ± 0.23  156 ±3 29 ± 1 ND 187 ± 5 H₂O₂ 47 ± 0.2* 41 ± 1.9* 2.44 ± 0.69*  27 ± 2*  6 ±1* 53 ± 6  36 ± 3* H₂O₂ + L- 27 ± 0.9 5 ± 2.0 2.71 ± 0.73* 151 ± 2  37 ±2** ND 189 ± 2 CAR P < 0.001, **P < 0.005, ND: not detectable

1. A method of preserving oxidant-protective activity ofalphaA-crystallin comprising contacting L-carnitine or a salt thereof toan alphaA-crystallin such that said alphaA-crystallin remains oxidantprotective, wherein said alphaA-crystallin is present in a nondiabetic,human patient.
 2. A method of preserving crystalline lens claritycomprising contacting L-carnitine or a salt thereof to said crystallinelens, wherein said crystalline lens is present in a nondiabetic, humanpatient.
 3. The method according to claim 1, wherein L-carnitine iscontacted to the alphaA-crystallin.
 4. The method according to claim 2,wherein L-carnitine is contacted to said crystalline lens.
 5. A methodof treating cataract in a nondiabetic, human patient comprisingadministering an ophthalmic composition comprised of a therapeuticallyeffective amount of L-carnitine or a salt thereof to an eye of saidnondiabetic, human patient.
 6. The method according to claim 5, whereinthe ophthalmic composition is comprised of a therapeutically effectiveamount of L-carnitine.
 7. The method according to claim 5, wherein saidtreatment preserves molecular chaperone activity of α-crystallin.
 8. Themethod according to claim 6, wherein said treatment preserves molecularchaperone activity of α-crystallin.
 9. The method according to claim 5,wherein said treatment preserves crystalline lens clarity when exposedto oxidative stress.
 10. The method according to claim 6, wherein saidtreatment preserves crystalline lens clarity when exposed to oxidativestress.
 11. The method according to claim 7, wherein said treatmentpreserves crystalline lens clarity when exposed to oxidative stress. 12.The method according to claim 8, wherein said treatment preservescrystalline lens clarity when exposed to oxidative stress.